Crystalline peptide epoxyketone immunoproteasome inhibitor

ABSTRACT

The invention relates to crystalline peptide keto epoxide compounds, methods of their preparation, and related pharmaceutical compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Stage application under 35 U.S.C. §371 ofInternational Application No. PCT/US2011/031436, having an internationalfiling date of Apr. 6, 2011, which claims priority to U.S. Provisionalapplication No. 61/321,577, filed Apr. 7, 2010, both applications areincorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

In eukaryotes, protein degradation is predominately mediated through theubiquitin pathway in which proteins targeted for destruction are ligatedto the 76 amino acid polypeptide ubiquitin. Once targeted, ubiquitinatedproteins then serve as substrates for the 26S proteasome, amulticatalytic protease, which cleaves proteins into short peptidesthrough the action of its three major proteolytic activities. Whilehaving a general function in intracellular protein turnover,proteasome-mediated degradation also plays a key role in many processessuch as major histocompatibility complex (MHC) class I presentation,apoptosis and cell viability, antigen processing, NF-κB activation, andtransduction of proinflammatory signals.

The 20S proteasome is a 700 kDa cylindrical-shaped multicatalyticprotease complex comprised of 28 subunits, classified as α- and β-type,that are arranged in 4 stacked heptameric rings. In yeast and othereukaryotes, 7 different α subunits form the outer rings and 7 differentβ subunits comprise the inner rings. The α subunits serve as bindingsites for the 19S (PA700) and 11S (PA28) regulatory complexes, as wellas a physical barrier for the inner proteolytic chamber formed by thetwo β subunit rings. Thus, in vivo, the proteasome is believed to existas a 26S particle (“the 26S proteasome”). In vivo experiments have shownthat inhibition of the 20S form of the proteasome can be readilycorrelated to inhibition of 26S proteasome.

Cleavage of amino-terminal prosequences of β subunits during particleformation expose amino-terminal threonine residues, which serve as thecatalytic nucleophiles. The subunits responsible for catalytic activityin proteasome thus possess an amino terminal nucleophilic residue, andthese subunits belong to the family of N-terminal nucleophile (Ntn)hydrolases (where the nucleophilic N-terminal residue is, for example,Cys, Ser, Thr, and other nucleophilic moieties). This family includes,for example, penicillin G acylase (PGA), penicillin V acylase (PVA),glutamine PRPP amidotransferase (GAT), and bacterialglycosylasparaginase. In addition to the ubiquitously expressed βsubunits, higher vertebrates also possess three interferon-γ-inducible βsubunits (LMP7, LMP2 and MECL1), which replace their normalcounterparts, β5, β1 and β2, respectively. When all threeIFN-γ-inducible subunits are present, the proteasome is referred to asan “immunoproteasome”. Thus, eukaryotic cells can possess two forms ofproteasomes in varying ratios.

Through the use of different peptide substrates, three major proteolyticactivities have been defined for the eukaryote 20S proteasomes:chymotrypsin-like activity (CT-L), which cleaves after large hydrophobicresidues; trypsin-like activity (T-L), which cleaves after basicresidues; and peptidylglutamyl peptide hydrolyzing activity (PGPH),which cleaves after acidic residues. Two additional less characterizedactivities have also been ascribed to the proteasome: BrAAP activity,which cleaves after branched-chain amino acids; and SNAAP activity,which cleaves after small neutral amino acids. Although both forms ofthe proteasome possess all five enzymatic activities, differences in theextent of the activities between the forms have been described based onspecific substrates. For both forms of the proteasome, the majorproteasome proteolytic activities appear to be contributed by differentcatalytic sites within the 20S core.

There are several examples of small molecules which have been used toinhibit proteasome activity; however, these compounds generally lack thespecificity to delineate between the two forms of the proteasome. Whatis needed are improved compositions and methods for preparing andformulating proteasome inhibitor(s).

SUMMARY OF THE INVENTION

One aspect of the invention relates to crystalline compounds having astructure of Formula (I) or a pharmaceutically acceptable salt thereof,

wherein

-   each Ar is independently an aromatic group optionally substituted    with 1 to 4 substituents;-   each A is independently selected from C═O, C═S, and SO₂; or-   A is optionally a covalent bond when adjacent to an occurrence of Z;-   B is absent or is N(R⁹)R¹⁰;-   L is absent or is selected from C═O, C═S, and SO₂;-   M is absent or is C₁₋₁₂alkyl;-   Q is absent or is selected from O, NH, and N—C₁₋₆alkyl;-   X is selected from O, S, NH, and N—C₁₋₆alkyl;-   Y is absent or is selected from C═O and SO₂;-   each Z is independently selected from O, S, NH, and N—C₁₋₆alkyl; or-   Z is optionally a covalent bond when adjacent to an occurrence of A;-   R¹ is selected from H, —C₁₋₆alkyl-B, C₁₋₆hydroxyalkyl,    C₁₋₆alkoxyalkyl, aryl, and C₁₋₆aralkyl;-   R² and R³ are each independently selected from aryl, C₁₋₆aralkyl,    heteroaryl, and C₁₋₆heteroaralkyl;-   R⁴ is N(R⁵)L-Q-R⁶;-   R⁵ is selected from hydrogen, OH, C₁₋₆aralkyl, and C₁₋₆alkyl;-   R⁶ is selected from hydrogen, C₁₋₆alkyl, C₁₋₆alkenyl, C₁₋₆alkynyl,    Ar—Y—, carbocyclyl, heterocyclyl, an N-terminal protecting group,    aryl, C₁₋₆aralkyl, R¹¹ZAZ—C₁₋₈alkyl-, R¹⁴Z—C₁₋₈alkyl-,    (R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-,    R¹¹ZAZ—C₁₋₈-alkyl-ZAZ—C₁₋₈alkyl-, heterocyclylMZAZ—C₁₋₈alkyl-,    (R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-, (R¹³)₂N—C₁₋₁₂alkyl-, heterocyclylM-,    carbocyclylM-, R¹⁴SO₂C₁₋₈alkyl-, and R¹⁴SO₂NH; or-   R⁵ and R⁶ together are C₁₋₆alkyl-Y—C₁₋₆alkyl,    C₁₋₆alkyl-ZAZ—C₁₋₆alkyl, ZAZ—C₁₋₆alkyl-ZAZ—C₁₋₆alkyl,    ZAZ—C₁₋₆alkyl-ZAZ, or C₁₋₆alkyl-A, thereby forming a ring;-   R⁷ and R⁸ are independently selected from hydrogen, C₁₋₆alkyl, and    C₁₋₆aralkyl;-   R⁹ is selected from hydrogen, OH, and C₁₋₆alkyl; and-   R¹⁰ is an N-terminal protecting group;-   R¹¹ and R¹² are independently selected from hydrogen, metal cation,    C₁₋₆alkyl, C₁₋₆alkenyl, C₁₋₆alkynyl, aryl, and C₁₋₆aralkyl;-   each R¹³ is independently selected from hydrogen and C₁₋₆alkyl; and-   R¹⁴ is independently selected from hydrogen, C₁₋₆alkyl, C₁₋₆alkenyl,    C₁₋₆alkynyl, carbocyclyl, heterocyclyl, aryl, and C₁₋₆aralkyl;-   R¹⁵ is selected from C₁₋₆alkyl, C₁₋₆hydroxyalkyl, C₁₋₆alkoxy,    —C(O)OC₁₋₆alkyl, —C(O)NHC₁₋₆alkyl, and C₁₋₆aralkyl;

provided that in any occurrence of the sequence ZAZ, at least one memberof the sequence must be other than a covalent bond.

Another aspect of the invention relates to a crystalline compound ofFormula (II)

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a DSC thermogram of the crystalline toluene solvate ofCompound 1.

FIG. 2 shows an XRPD of the crystalline toluene solvate of Compound 1.

FIG. 3 shows a TG thermogram of crystalline toluene solvate of Compound1.

FIG. 4 shows an ORTEP diagram of a single molecule of the of crystallinetoluene solvate of Compound 1.

FIG. 5 shows a DSC thermogram of an amorphous form of Compound 1.

FIG. 6 shows an XRPD pattern of an amorphous form of Compound 1.

DETAILED DESCRIPTION OF THE INVENTION

In certain embodiments, the invention relates to crystalline compoundshaving a structure of Formula (I) or a pharmaceutically acceptable saltthereof,

wherein

-   each Ar is independently an aromatic group optionally substituted    with 1 to 4 substituents;-   each A is independently selected from C═O, C═S, and SO₂; or-   A is optionally a covalent bond when adjacent to an occurrence of Z;-   B is absent or is N(R⁹)R¹⁰;-   L is absent or is selected from C═O, C═S, and SO₂;-   M is absent or is C₁₋₁₂alkyl;-   Q is absent or is selected from O, NH, and N—C₁₋₆alkyl;-   X is selected from O, S, NH, and N—C₁₋₆alkyl;-   Y is absent or is selected from C═O and SO₂;-   each Z is independently selected from O, S, NH, and N—C₁₋₆alkyl; or-   Z is optionally a covalent bond when adjacent to an occurrence of A;-   R¹ is selected from H, —C₁₋₆alkyl-B, C₁₋₆hydroxyalkyl,    C₁₋₆alkoxyalkyl, aryl, and C₁₋₆aralkyl;-   R² and R³ are each independently selected from aryl, C₁₋₆aralkyl,    heteroaryl, and C₁₋₆heteroaralkyl;-   R⁴ is N(R⁵)L-Q-R⁶;-   R⁵ is selected from hydrogen, OH, C₁₋₆aralkyl, and C₁₋₆alkyl;-   R⁶ is selected from hydrogen, C₁₋₆alkyl, C₁₋₆alkenyl, C₁₋₆alkynyl,    Ar—Y—, carbocyclyl, heterocyclyl, an N-terminal protecting group,    aryl, C₁₋₆aralkyl, R¹¹ZAZ—C₁₋₈alkyl-, R¹⁴Z—C₁₋₈alkyl-,    (R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-,    R¹¹ZAZ—C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-, heterocyclylMZAZ—C₁₋₈alkyl-,    (R¹¹O)(R¹²O)P(═O)O—C₁₋₈alkyl-, (R¹³)₂N—C₁₋₁₂alkyl-,    (R¹³)₃N—C₁₋₁₂alkyl-, heterocyclylM-, carbocyclylM-,    R¹⁴SO₂C₁₋₈alkyl-, and R¹⁴SO₂NH; or-   R⁵ and R⁶ together are C₁₋₆alkyl-Y—C₁₋₆alkyl,    C₁₋₆alkyl-ZAZ—C₁₋₆alkyl, ZAZ—C₁₋₆alkyl-ZAZ—C₁₋₆alkyl,    ZAZ—C₁₋₆alkyl-ZAZ, or C₁₋₆alkyl-A, thereby forming a ring;-   R⁷ and R⁸ are independently selected from hydrogen, C₁₋₆alkyl, and    C₁₋₆aralkyl;-   R⁹ is selected from hydrogen, OH, and C₁₋₆alkyl; and-   R¹⁰ is an N-terminal protecting group;-   R¹¹ and R¹² are independently selected from hydrogen, metal cation,    C₁₋₆alkyl, C₁₋₆alkenyl, C₁₋₆alkynyl, aryl, and C₁₋₆aralkyl;-   each R¹³ is independently selected from hydrogen and C₁₋₆alkyl;-   R¹⁴ is independently selected from hydrogen, C₁₋₆alkyl, C₁₋₆alkenyl,    C₁₋₆alkynyl, carbocyclyl, heterocyclyl, aryl, and C₁₋₆aralkyl; and-   R¹⁵ is selected from C₁₋₆alkyl, C₁₋₆hydroxyalkyl, C₁₋₆alkoxy,    —C(O)OC₁₋₆alkyl, —C(O)NHC₁₋₆alkyl, and C₁₋₆aralkyl;

provided that in any occurrence of the sequence ZAZ, at least one memberof the sequence must be other than a covalent bond.

In certain embodiments, R⁷ and R⁸ are independently selected fromhydrogen and C₁₋₆alkyl, preferably R⁷ and R⁸ are both hydrogen.

In certain embodiments, R¹⁵ is selected from C₁₋₆alkyl andC₁₋₆hydroxyalkyl. In certain such embodiments, R¹⁵ is selected frommethyl, ethyl, hydroxymethyl, and 2-hydroxyethyl, preferably methyl.

In certain embodiments, R⁵ is hydrogen or C₁₋₆alkyl, preferably R⁵ ishydrogen.

In certain embodiments, R¹ is selected from —C₁₋₆alkylB and C₁₋₆aralkyl.In certain preferred embodiments, R¹ is selected from methyl, ethyl,isopropyl, carboxymethyl, and benzyl, preferably methyl.

In certain embodiments, R² is selected from C₁₋₆aralkyl andC₁₋₆heteroaralkyl. In certain such embodiments, R² is selected fromC₁₋₆alkyl-phenyl, C₁₋₆alkyl-indolyl, C₁₋₆alkyl-thienyl,C₁₋₆alkyl-thiazolyl, and C₁₋₆alkyl-isothiazolyl, preferablyC₁₋₆alkyl-phenyl.

In certain embodiments, R² is selected from

wherein D is selected from hydrogen, methoxy, t-butoxy, hydroxy, cyano,trifluoromethyl, and C₁₋₄alkyl; and R is hydrogen or a suitableprotecting group.

In certain embodiments, R³ is selected from C₁₋₆aralkyl andC₁₋₆heteroaralkyl.

In certain embodiments, R³ is selected from C₁₋₆alkyl-phenyl andC₁₋₆alkyl-indolyl, preferably C₁₋₆alkyl-phenyl.

In certain embodiments, R³ is selected from

wherein D is selected from hydrogen, methoxy, t-butoxy, hydroxy, cyano,trifluoromethyl, and C₁₋₄alkyl; and R is hydrogen or a suitableprotecting group.

In certain embodiments, L is C═O, Q is absent, and R⁶ is selected fromheterocyclyl and heterocyclylM-, preferably heterocyclylM-. In certainsuch embodiments, R⁶ is heterocyclylM-, where heterocyclyl is selectedfrom morpholino, piperidino, piperazino, and pyrrolidino, preferablymorpholino. In certain embodiments, R⁶ is morpholinomethyl.

In certain embodiments, the invention relates to a crystalline compoundof Formula (II)

In certain embodiments, the invention relates to a method for thepreparation of a crystalline compound of Formula (I) or (II), comprisingone or more of: (i) preparing the amorphous compound, e.g., according toU.S. patent application Ser. No. 11/820,490; (ii) dissolving theamorphous compound in an organic solvent; (iii) bringing the solution tosupersaturation to cause formation of crystals; and (iv) isolating thecrystals, e.g., by filtering the crystals, by decanting fluid from thecrystals, or by any other suitable separation technique. In certainembodiments, preparation further comprises inducing crystallization. Incertain embodiments, preparation further comprises washing the filteredcrystals, e.g., with a solvent or non-solvent fluid. In certainembodiments, preparation further comprises drying, preferably underreduced pressure, such as under vacuum pressure.

In certain embodiments, the invention relates to a method for thepreparation of a crystalline compound of Formula (I) or (II), comprisingone or more of: (i) preparing a solution of the amorphous compound,which compound may be prepared according to, for example, U.S. patentapplication Ser. No. 11/820,490, in an organic solvent; (ii) bringingthe solution to supersaturation to cause formation of crystals; and(iii) isolating the crystals, e.g., by filtering the crystals, bydecanting fluid from the crystals, or by any other suitable separationtechnique. In certain embodiments, preparation further comprisesinducing crystallization. In certain embodiments, preparation furthercomprises washing the filtered crystals, e.g., with a solvent ornon-solvent fluid. In certain embodiments, preparation further comprisesdrying, preferably under reduced pressure, such as under vacuumpressure.

In certain embodiments, the amorphous compound may be dissolved in anorganic solvent selected from acetonitrile, acetone, diethyl ether,diisopropyl ether, ethanol, ethyl acetate, heptanes, isopropyl acetate,methanol, methyl tert-butyl ether, tetrahydrofuran, toluene, heptanes,or any combination thereof. In certain embodiments, the amorphouscompound may be dissolved in an organic solvent selected fromacetonitrile, acetone, diethyl ether, diisopropyl ether, ethyl acetate,heptanes, isopropyl acetate, methanol, methyl tert-butyl ether,tetrahydrofuran, toluene, heptanes, or any combination thereof. Incertain embodiments, the amorphous compound may be dissolved in anorganic solvent selected from diethyl ether, diisopropyl ether, methyltert-butyl ether, tetrahydrofuran, toluene, heptanes, or any combinationthereof. In certain embodiments, the amorphous compound may be dissolvedin toluene, methyl tert-butyl ether, heptanes, or a combination thereof.In certain preferred embodiments, the amorphous compound may bedissolved in toluene.

In certain embodiments, bringing the solution to supersaturationcomprises the addition of an anti-solvent, such as another liquidmiscible with the organic solvent, allowing the solution to cool,reducing the volume of the solution, or any combination thereof. Incertain embodiments, bringing the solution to supersaturation comprisesadding an anti-solvent, cooling the solution to ambient temperature orlower, and reducing the volume of the solution, e.g., by evaporatingsolvent from the solution. In certain embodiments, allowing the solutionto cool may be passive (e.g., allowing the solution to stand at ambienttemperature) or active (e.g., cooling the solution in an ice bath orfreezer).

In certain embodiments, the method further comprises inducingprecipitation or crystallization. In certain embodiments inducingprecipitation or crystallization comprises secondary nucleation, whereinnucleation occurs in the presence of seed crystals or interactions withthe environment (crystallizer walls, stirring impellers, sonication,etc.).

In certain embodiments, washing the crystals comprises washing with aliquid selected from anti-solvent, acetonitrile, acetone, diethyl ether,diisopropyl ether, ethanol, ethyl acetate, heptanes, isopropyl acetate,methanol, methyl tert-butyl ether, toluene, tetrahydrofuran, or anycombination thereof. In certain embodiments, the crystals are washedwith an organic solvent selected from acetonitrile, acetone, diethylether, diisopropyl ether, ethyl acetate, heptanes, isopropyl acetate,methanol, methyl tert-butyl ether, tetrahydrofuran, toluene, or anycombination thereof. In certain embodiments, the crystals are washedwith toluene, methyl tert-butyl ether, heptanes, or a combinationthereof.

In certain embodiments, washing the crystals comprises washing thecrystalline compound of Formula (II) with toluene or toluene incombination with methyl tert-butyl ether.

In certain embodiments, the DSC of a crystalline compound of Formula(II) has a sharp endothermic maximum at about 83° C. (onset at about 78°C., heating rate of 10° C./min) resulting from melting and decompositionof the crystalline form as shown in FIG. 1.

In certain embodiments, the X-ray powder pattern of a crystallinecompound of Formula (II) is (θ-2θ°): 3.62±0.20; 6.52±0.20; 7.20±0.20;8.66±0.20; 10.04±0.20; 10.82±0.20; 12.98±0.20; 14.42±0.20; 16.46±0.20;17.32±0.20; 17.98±0.20; 18.02±0.20; 18.88±0.20; 19.06±0.20; 19.56±0.20;19.88±0.20; 20.40±0.20; 21.36±0.20; 22.16±0.20; 23.20±0.20; 25.40±0.20;28.82±0.20 as shown in FIG. 2.

In certain embodiments, the TG thermogram of a crystalline compound ofFormula (II) exhibits from 5.6% weight losses up to 150° C. as shown inFIG. 3.

In certain embodiments, a crystalline compound of Formula (II) is notsolvated (e.g., the crystal lattice does not comprise molecules of asolvent). In certain alternative embodiments, a crystalline compound ofFormula (II) is solvated. In certain preferred embodiments, acrystalline compound of Formula (II) is a toluene solvate.

In certain embodiments, the invention relates to a method for thepreparation of an amorphous compound of Formula (II) comprising one ormore of (i) dissolving the crystalline compound in an organic solvent;(ii) bringing the solution to supersaturation to cause formation ofcrystals; and (iii) isolating the crystals, e.g., by filtering thecrystals, by decanting fluid from the crystals, or by any other suitableseparation technique. In certain embodiments, preparation furthercomprises inducing precipitation. In certain embodiments, preparationfurther comprises washing the amorphous compound. In certainembodiments, the method further comprises drying, preferably underreduced pressure, such as under vacuum pressure.

In certain embodiments, the invention relates to a pharmaceuticalcomposition comprising a crystalline compound of Formula (I) or (II) anda pharmaceutically acceptable carrier. In certain embodiments, thepharmaceutical composition is selected from tablets, capsules, andinjections.

Uses of Enzyme Inhibitors

The biological consequences of proteasome inhibition are numerous.Proteasome inhibition has been suggested as a prevention and/ortreatment of a multitude of diseases including, but not limited to,proliferative diseases, neurotoxic/degenerative diseases, ischemicconditions, inflammation, immune-related diseases, HIV, cancers, organgraft rejection, septic shock, viral and parasitic infections,conditions associated with acidosis, macular degeneration, pulmonaryconditions, muscle wasting diseases, fibrotic diseases, bone and hairgrowth diseases.

Proteasome inhibitors can be used to treat conditions mediated directlyby the proteolytic function of the proteasome such as muscle wasting, ormediated indirectly via proteins which are processed by the proteasomesuch as NF-κB. The proteasome participates in the rapid elimination andpost-translational processing of proteins (e.g., enzymes) involved incellular regulation (e.g., cell cycle, gene transcription, and metabolicpathways), intercellular communication, and the immune response (e.g.,antigen presentation).

At the cellular level, the accumulation of polyubiquitinated proteins,cell morphological changes, and apoptosis have been reported upontreatment of cells with various proteasome inhibitors. Yet, it should benoted that commercially available proteasome inhibitors inhibit both theconstitutive and immuno forms of the proteasome. Even bortezomib, theonly FDA-approved proteasome inhibitor for the treatment of relapsedmultiple myeloma patients, does not distinguish between the two forms(Altun et al., Cancer Res 65:7896, 2005). Thus, what is known abouttherapeutic proteasome inhibition is based on work with molecules thatinhibit both forms of the proteasome. Accordingly, compounds of theinvention may be beneficial for reducing the severity of side effectsassociated with molecules that inhibit both forms of the proteasome.

Immunoproteasome expression occurs predominantly in cells and organsthat make up the lymphatic system, such as white blood cells(leukocytes), bone marrow, and the thymus, spleen and lymph nodes.Although some organs preferentially express constitutive proteasomes(e.g., heart), others such as adrenal, liver, lung and gut, appear toexpress both forms.

The immune system, of which leukocytes and lymphoid tissues play a majorrole, is responsible for protecting an organism from outside biologicalinfluences. When functioning properly, it protects the body againstbacterial and viral infections. The immune system also screens forautologous cells that have undergone oncogenic transformation.Intracellular proteolysis generates small peptides for presentation toT-lymphocytes to induce MHC class I-mediated immune responses. Theproteasome is the main provider of these precursor peptides; however,differences between antigenic peptides have been observed between cellswith varying amounts of each proteasome form (Cascio et al., EMBO J.20:2357-2366, 2001). In certain embodiments, the invention relates to amethod for inhibiting antigen presentation in a cell, including exposingthe cell to a compound as described herein. In certain embodiments, theinvention relates to a method for altering the repertoire of antigenicpeptides produced by the proteasome or other Ntn with multicatalyticactivity. For example, if the activity of the immunoproteasomeproteasome is selectively inhibited, a different set of antigenicpeptides may be produced by the remaining constitutive proteasome andpresented in MHC molecules on the surfaces of cells than would beproduced and presented without any enzyme inhibition.

Several disorders and disease states have been associated with aberrantimmune system function, herein referred to as immune-related conditions.Perhaps the most common immune-related condition is the allergicdisorders such as allergies, asthma and atopic dermatitis like eczema.These occur when the immune system overreacts to exposure to antigens inthe environment. Thus, a further embodiment is a method for suppressingthe immune system of a subject, comprising administering to the subjectan effective amount of a proteasome inhibitor compound in a mannerdescribed herein.

Immunodeficiency disorders occur when a part of the immune system is notworking properly or is not present. They can affect B lymphocytes, Tlymphocytes, or phagocytes and be either inherited (e.g., IgAdeficiency, severe combined immunodeficiency (SCID), thymic dysplasiaand chronic granulomatous) or acquired (e.g., acquired immunodeficiencysyndrome (AIDS), human immunodeficiency virus (HIV) and drug-inducedimmunodeficiencies). A dosing strategy utilizing selective proteasomeinhibitors of the invention may be used to treat immune-relatedconditions such as immunodeficiency disorders.

In autoimmune disorders, the immune system inappropriately attacks thebody's healthy organs and tissues as if they were foreign invaders. Anexample of an autoimmune disease is Sjogren's Syndrome, which ischaracterized by infiltration and focal accumulation of lymphocytes inthe exocrine glands. A study examining the proteasome expression levelrevealed a significant up-regulation of beta5i (LMP7) exclusively in thesalivary glands of SS patients (Egerer et al., Arthritis Rheum54:1501-8, 2006). Other examples of such immune-related conditionsinclude lupus (such as lupus nephritis and systemic lupuserythamotosus), rheumatoid arthritis (such as juvenile rheumatoidarthritis and psoriatic rheumatoid arthritis), scleroderma, ankylosingspondylitis, dermatomyositis, psoriasis, multiple sclerosis (bothrelapsing remitting and chronic progressive forms) and inflammatorybowel disease (such as ulcerative colitis and Crohn's disease).Tissue/organ transplant rejection occurs when the immune systemmistakenly attacks the cells being introduced to the host's body. Graftversus host disease (GVHD), resulting from allogenic transplantation,arises when the T cells from the donor tissue go on the offensive andattack the host's tissues. In all three circumstances, autoimmunedisease, transplant rejection and GVHD, modulating the immune system bytreating the subject with a compound of the invention could bebeneficial.

Inflammation is the first response of the immune system to infection orirritation. A cellular component of inflammation involves the movementof leukocytes, which express immunoproteasome, from blood vessels intothe inflamed tissue. These cells take on the important role of removingthe irritant, bacteria, parasite or cell debris. Proteasome inhibitorsare already known to have anti-inflammatory activity (Meng et al., PNAS96:10403-10408, 1999). In cases of chronic inflammation, which ischaracterized by a dominating presence of macrophages, the cells thatoriginally served as defensive agents begin to release toxins andcytokines, including TNF-α, now become injurious to the body, resultingin tissue damage and loss. In certain embodiments, the invention relatesto a method of treating inflammation and inflammatory diseasescomprising administering a compound as described herein. Inflammatorydiseases include acute (e.g., bronchitis, conjunctivitis, pancreatitis)and chronic conditions (e.g., chronic cholecstitis, bronchiectasis,aortic valve stenosis, restenosis, psoriasis and arthritis), along withconditions associated with inflammation such as fibrosis, infection andischemia. In certain embodiments, the invention relates to methods oftreating conditions whereby an organ is inflamed, such as autoimmunemyocarditis and autoimmune thyroiditis.

Following tissue damage, including damage due to the inflammationprocess, progression of regeneration and repair begins. During theregeneration step, lost tissue is replaced by proliferation of cells ofthe same type, which reconstruct the normal architecture. However,improper regeneration of the tissue architecture may have severeconsequences. In some cases of chronic inflammatory liver disease, theregenerated tissue forms an abnormal nodular architecture leading tocirrhosis and portal hypertension. The repair process results in losttissue being replaced by a fibrous scar which is produced fromgranulation tissue. Fibrosis is the excessive and persistent formationof scar tissue resulting from the hyperproliferative growth offibroblasts and is associated with activation of the TGF-β signalingpathway. Fibrosis involves extensive deposition of extracellular matrixand can occur within virtually any tissue or across several differenttissues. Normally, the level of intracellular signaling protein (Smad)that activate transcription of target genes upon TGF-β stimulation isregulated by proteasome activity (Xu et al., 2000). However, accelerateddegradation of the TGF-β signaling components has been observed incancers and other hyperproliferative conditions. Thus, in certainembodiments, the invention relates to a method for treating ahyperproliferative condition selected from diabetes (such as type 1,type 2 and metabolic syndrome), diabetic retinopathy, maculardegeneration, diabetic nephropathy, glomerulosclerosis, IgA nephropathy,cirrhosis, biliary atresia, congestive heart failure, scleroderma,radiation-induced fibrosis, and lung fibrosis (idiopathic pulmonaryfibrosis, collagen vascular disease, sarcoidosis, interstitial lungdiseases and extrinsic lung disorders), comprising administering acompound as described herein. The treatment of burn victims is oftenhampered by fibrosis, thus, in certain embodiments, the inventionrelates to the topical or systemic administration of a compound asdescribed herein to treat burns. Wound closure following surgery isoften associated with disfiguring scars, which may be prevented byinhibition of fibrosis. Thus, in certain embodiments, the inventionrelates to a method for the prevention or reduction of scarring,comprising administering a compound as described herein.

Infection by bacteria, parasite or virus all result in initiation of theinflammatory process. When the resulting inflammation overwhelms thewhole organism, systemic inflammatory response syndrome (SIRS) occurs.The term sepsis is applied when this is due to infection. Overproductionof lipopolysaccharide (LPS)-induced cytokines such as TNFα is consideredto be central to the processes associated with septic shock. Notsurprisingly, LPS also induces an increase in all components of theMHC-1 pathway including the immunoproteasome subunits LMP2 and LMP7(MacAry et al., PNAS 98:3982-3987, 2001). Furthermore, it is generallyaccepted that the first step in the activation of cells by LPS is thebinding of LPS to specific membrane receptors. The α- and β-subunits ofthe 20S proteasome complex have been identified as LPS-binding proteins,suggesting that the LPS-induced signal transduction may be an importanttherapeutic target in the treatment or prevention of sepsis (Qureshi, N.et al., J. Immun. (2003) 171: 1515-1525). Therefore, in certainembodiments, the invention relates to preventing or treating septicshock, comprising administering a compound as disclosed herein.

In another embodiment, the disclosed compositions are useful for thetreatment of a parasitic infection, such as infections caused byprotozoan parasites. The proteasome of these parasites is considered tobe involved primarily in cell differentiation and replication activities(Paugam et al., Trends Parasitol. 2003, 19(2): 55-59). Furthermore,entamoeba species have been shown to lose encystation capacity whenexposed to proteasome inhibitors (Gonzales, et al., Arch. Med. Res.1997, 28, Spec No: 139-140). In certain such embodiments, the compoundsas disclosed herein are useful for the treatment of parasitic infectionsin humans caused by a protozoan parasite selected from Plasmodium sps.(including P. falciparum, P. vivax, P. malariae, and P. ovale, whichcause malaria), Trypanosoma sps. (including T. cruzi, which causesChagas' disease, and T. brucei which causes African sleeping sickness),Leishmania sps. (including L. amazonesis, L. donovani, L. infantum, L.mexicana, etc.), Pneumocystis carinii (a protozoan known to causepneumonia in AIDS and other immunosuppressed patients), Toxoplasmagondii, Entamoeba histolytica, Entamoeba invadens, and Giardia lamblia.In certain embodiments, the disclosed compositions are useful for thetreatment of parasitic infections in animals and livestock caused by aprotozoan parasite selected from Plasmodium hermani, Cryptosporidiumsps., Echinococcus granulosus, Eimeria tenella, Sarcocystis neurona, andNeurospora crassa. Other compounds useful as proteasome inhibitors inthe treatment of parasitic diseases are described in WO 98/10779, whichis incorporated herein by reference in its entirety.

In certain embodiments, compounds disclosed herein may inhibitproteasome activity in a parasite without recovery in white blood cells.In certain such embodiments, the long half-life of blood cells mayprovide prolonged protection with regard to therapy against recurringexposures to parasites. In certain embodiments, the compounds asdescribed herein may provide prolonged protection with regard tochemoprophylaxis against future infection.

Viral infections contribute to the pathology of many diseases. Heartconditions such as ongoing myocarditis and dilated cardiomyopathy havebeen linked to the coxsackievirus B3. In a comparative whole-genomemicroarray analyses of infected mouse hearts, all three immunoproteasomesubunits were uniformly up-regulated in hearts of mice which developedchronic myocarditis (Szalay et al., Am J Pathol 168:1542-52, 2006). Someviruses utilize the ubiquitin-proteasome system in the viral entry stepwhere the virus is released from the endosome into the cytosol. Themouse hepatitis virus (MHV) belongs to the Coronaviridae family, whichalso includes the severe acute respiratory syndrome (SARS) coronvirus.Yu and Lai (J Virol 79:644-648, 2005) demonstrated that treatment ofcells infected with MHV with a proteasome inhibitor resulted in adecrease in viral replication, correlating with reduced viral titer ascompared to that of untreated cells. The human hepatitis B virus (HBV),a member of the Hepadnaviridae virus family, requires virally encodedenvelop proteins to propagate. Inhibiting the proteasome degradationpathway causes a significant reduction in the amount of secretedenvelope proteins (Simsek et al., J Virol 79:12914-12920, 2005). Inaddition to HBV, other hepatitis viruses (A, C, D and E) may alsoutilize the ubiquitin-proteasome degradation pathway for secretion,morphogenesis and pathogenesis. Accordingly, in certain embodiments theinvention relates to treating a viral infection, comprisingadministering a compound as disclosed herein.

The bacterium Listeria monocytogenes causes a condition known aslisteriosis, the manifestations of which range from mild (nausea,vomiting and diarrhea) to severe (septicemia, meningitis, encephalitis).A quantitative analysis of changes in proteasome subunit compositionrevealed that infection of mice with lymphocytic choriomeningitis virusor Listeria monocytogenes lead to an almost complete replacement ofconstitutive proteasomes by immunoproteasomes in the liver within sevendays (Khan et al., J Immunol 167:6859-6868, 2001). Prokaryotes have whatis equivalent to the eukaryote 20S proteasome particle. While thesubunit composition of the prokaryote 20S particle is simpler than thatof eukaryotes, it does have the ability to hydrolyze peptide bonds in asimilar manner. For example, the nucleophilic attack on the peptide bondoccurs through the threonine residue on the N-terminus of theβ-subunits. Thus, in certain embodiments, the invention relates to amethod of treating prokaryotic infections, comprising administering acompound as disclosed herein. Prokaryotic infections may includediseases caused by either mycobacteria (such as tuberculosis, leprosy orBuruli Ulcer) or archaebacteria.

In certain embodiments, the invention relates to a method for treatinginfection (e.g., bacterial, parasitic or viral), comprising contacting acell with a compound as disclosed herein. In certain alternativeembodiments, the invention relates to a method for treating infection,comprising administering a compound as disclosed herein.

Ischemia and reperfusion injury results in hypoxia, a condition in whichthere is a deficiency of oxygen reaching the tissues of the body. Thiscondition causes increased degradation of Iκ-Bα, thereby resulting inthe activation of NF-κB (Koong et al., 1994). Interestingly, factorswhich have been identified as being able to enhance immunoproteasomeexpression, TNF-α and lipopolysaccharide, also stimulate NF-κBactivation. It has been demonstrated that the severity of injuryresulting in hypoxia can be reduced with the administration of aproteasome inhibitor (Gao et al., 2000; Bao et al., 2001; Pye et al.,2003). Therefore, in certain embodiments the invention relates to amethod of treating an ischemic condition or reperfusion injury,comprising administering a compound as disclosed herein. Examples ofsuch conditions or injuries include, but are not limited to, acutecoronary syndrome (vulnerable plaques), arterial occlusive disease(cardiac, cerebral, peripheral arterial and vascular occlusions),atherosclerosis (coronary sclerosis, coronary artery disease),infarctions, heart failure, pancreatitis, myocardial hypertrophy,stenosis, and restenosis.

Cachexia is a syndrome characterized by wasting of skeletal muscleassociated with enhanced proteolysis due to the ubiquitin-proteasomepathway. Inhibiting the proteasome reduces proteolysis, thereby reducingboth muscle protein loss and the nitrogenous load on kidneys or liver(Tawa et al., JCI 100:197-203, 1997). In cachexia, elevated expressionof proinflammatory cytokines, TNF-α and IFN-γ, both of which stimulateexpression of immunoproteasome subunits, have been reported (Acharyya etal., JCI 114:370-378, 2004). In fact, most types of muscle atrophyexhibit elevated rates of protein degradation (Lecker et al., FASEB J18:39-51, 2004). Muscle wasting manifests itself in several lifethreatening diseases, including cancer, sepsis, renal failure, AIDS,fasting, denervation atrophy, acidosis, diabetes, disuse atrophy andcongestive heart failure. In certain embodiments, the invention relatesto the treatment of cachexia or muscle-wasting disease, comprisingadministering a compound as disclosed herein. Compounds of the inventionmay be useful for treating conditions such as cancer, chronic infectiousdiseases, fever, muscle disuse (atrophy) and denervation, nerve injury,fasting, renal failure associated with acidosis, and hepatic failure.See, e.g., Goldberg, U.S. Pat. No. 5,340,736.

Degradation of certain proteins by the proteasome effect signalingmechanisms that, in turn, effect gene transcription, cell cycle andmetabolic pathways. As noted above, proteasome inhibitors block bothdegradation and processing of ubiquitinated NF-κB in vitro and in vivo.Proteasome inhibitors also block IκB-A degradation and NF-κB activation(Palombella et al., Cell (1994) 78:773-785; and Traenckner et al., EMBOJ. (1994) 13:5433-5441). In certain embodiments, the invention relatesto a method for inhibiting IκB-α degradation, comprising contacting thecell with a compound as described herein.

In certain embodiments, the invention relates to methods for affectingcyclin-dependent eukaryotic cell cycles, including exposing a cell (invitro or in vivo) to a compound as disclosed herein. Cyclins areproteins involved in cell cycle control. The proteasome participates inthe degradation of cyclins. Examples of cyclins include mitotic cyclins,G1 cyclins, and cyclin B. Degradation of cyclins enables a cell to exitone cell cycle stage (e.g., mitosis) and enter another (e.g., division).It is believed all cyclins are associated with p34^(cdc2) protein kinaseor related kinases. The proteolysis targeting signal is localized toamino acids 42-50 (destruction box). There is evidence that cyclin isconverted to a form vulnerable to a ubiquitin ligase or that acyclin-specific ligase is activated during mitosis (Ciechanover, A.,Cell, (1994) 79:13-21). Inhibition of the proteasome inhibits cyclindegradation, and therefore inhibits cell proliferation, for example, incyclin-related cancers (Kumatori et al., Proc. Natl. Acad. Sci. USA(1990) 87:7071-7075). In certain embodiments, the invention relates to amethod for treating a proliferative disease in a subject (e.g., cancer,psoriasis, or restenosis), comprising administering a compound asdisclosed herein. In certain embodiments, the invention also relates toa method for treating cyclin-related inflammation, comprisingadministering a compound as described herein.

In maturing reticulocytes and growing fibroblasts, or cells deprived ofinsulin or serum, the rate of proteolysis nearly doubles, suggesting arole for the proteasome in cellular metabolism. Accordingly, in certainembodiments, the invention relates to methods for reducing the rate ofintracellular protein degradation in a cell. Each of these methodscomprises contacting a cell (in vivo or in vitro) with a compound asdisclosed herein.

Alzheimer's disease (AD) is a progressive neurodegenerative diseasedisorder associated with a loss of higher cognitive function.Pathological hallmarks of the disease include senile amyloid plaques,neurofibrillary tangles, dystrophic neuritis and significant neuronalloss in selected regions of the brain. Microglia, the residentmacrophages in the brain, release numerous proinflammatory cytokines,including TNF-α, when activated by Aβ42, a peptide associated withneuritic and vascular amyloid plaques. This microglial-mediatedinflammatory response contributes to significant neuronal loss.Cell-based studies demonstrated that primary cortical neurons treatedwith conditioned media from microglial BV2 cells stimulated either withLPS/INF-γ or sonicated Aβ42 peptides resulted in approximately a 60%decrease in cell viability (Gan et al., J. Biol. Chem. 279:5565-5572,2004). A higher expression of immunoproteasome is found in brain tissuefrom AD patients than in that of non-demented elderly adults (Mishto etal., Neurobiol Aging 27:54-66, 2006).

Patients suffering from Huntington's disease (HD), anotherneurodegenerative disorder, display motor dysfunction and cognitivedecline over a period of years until death. Upon autopsy, the presenceof inclusions or intraneuronal aggregates, caused by a polyQ expansionmutation (also referred to as a CAG triplet repeat expansion), can bedetected, accompanied by significant atrophy in the striatum and cortexportions of the brain. Immunohistochemistry revealed that there was asignificant enhancement in immunoproteasome expression in the striatumand frontal cortex of brains from HD patients as compared to those fromage-matched normal adults (Diaz-Hernandez et al., J Neurosci23:11653-1161, 2003). Upon further analysis, it was discovered that theenhancement predominantly occurred in the degenerating neurons. Using amouse model of HD, the researchers noted a selective increase in bothchymotrypsin- and trypsin-like activities in the affected andaggregate-containing regions of the brain, primarily the cortex andstriatum (Diaz-Hernandez et al, J Neurosci 23:11653-1161, 2003).

Accordingly, certain embodiments of the invention relate to a method fortreating a neurodegenerative disease or condition, comprisingadministering a compound as disclosed herein. Neurodegenerative diseasesand conditions include, but are not limited to, stroke, ischemic damageto the nervous system, neural trauma (e.g., percussive brain damage,spinal cord injury, and traumatic damage to the nervous system),multiple sclerosis and other immune-mediated neuropathies (e.g.,Guillain-Barre syndrome and its variants, acute motor axonal neuropathy,acute inflammatory demyelinating polyneuropathy, and Fisher Syndrome),HIV/AIDS dementia complex, axonomy, diabetic neuropathy, Parkinson'sdisease, Huntington's disease, multiple sclerosis, bacterial, parasitic,fungal, and viral meningitis, encephalitis, vascular dementia,multi-infarct dementia, Lewy body dementia, frontal lobe dementia suchas Pick's disease, subcortical dementias (such as Huntington orprogressive supranuclear palsy), focal cortical atrophy syndromes (suchas primary aphasia), metabolic-toxic dementias (such as chronichypothyroidism or B12 deficiency), and dementias caused by infections(such as syphilis or chronic meningitis).

It has also been demonstrated that inhibitors that bind to the 20Sproteasome stimulate bone formation in bone organ cultures. Furthermore,when such inhibitors have been administered systemically to mice,certain proteasome inhibitors increased bone volume and bone formationrates over 70% (Garrett, I. R. et al., J. Clin. Invest. (2003) 111:1771-1782), therefore suggesting that the ubiquitin-proteasome machineryregulates osteoblast differentiation and bone formation. Therefore, thedisclosed proteasome inhibitor compositions may be useful in thetreatment and/or prevention of diseases associated with bone loss, suchas osteoporosis.

Cancer is a general term for disease characterized by uncontrolled,abnormal growth of cells. Many cancers arise via multistep pathwaysinvolving inactivation of tumor suppressor proteins and activation ofoncogenic peptides. Cancer cells can spread to other parts of the bodythrough the lymphatic system or blood stream. Usually, cancer isclassified according to the type of tissue or cell most prominentlyinvolved. As noted previously, proteasome inhibition has already beenvalidated as a therapeutic strategy for the treatment of cancer,particularly multiple myeloma. Multiple myeloma cells possess both formsof the proteasome, although the ratio can vary somewhat. Multiplemyeloma is a hematologic disease characterized by an excessive number ofabnormal plasma cells in the bone marrow. Plasma cells develop fromB-cells, thus it is not surprising that other B-cell malignancies wouldalso express immunoproteasome to some extent. Except for two chronicmylogenous leukemia cell lines, heme-related cancers (e.g., multiplemyeloma, leukemias and lymphomas) generally appear to expressimmunoproteasome. Cancer cells originating from lymphoid cells express30% or more immunoproteasome. In certain embodiments, the inventionrelates to a method for the treatment of cancer, comprisingadministering a compound as described herein. In certain preferredembodiments, the cancer is a heme-related disorder. In certainembodiments, the cancer is selected from a solid tumor, head and necksquamous cell carcinoma, cervical carcinoma and small cell lungcarcinoma.

The treatment of the cells with INF-γ can induce immunoproteasomeexpression. Therefore, in certain embodiments, the invention relates toa method of treating cancer comprising administering a compound asdisclosed herein.

Administration

Compounds prepared as described herein can be administered in variousforms, depending on the disorder to be treated and the age, condition,and body weight of the patient, as is well known in the art. Forexample, where the compounds are to be administered orally, they may beformulated as tablets, capsules, granules, powders, or syrups; or forparenteral administration, they may be formulated as injections(intravenous, intramuscular, or subcutaneous), drop infusionpreparations, or suppositories. For application by the ophthalmic mucousmembrane route, they may be formulated as eye drops or eye ointments.These formulations can be prepared by conventional means, and ifdesired, the active ingredient may be mixed with any conventionaladditive or excipient, such as a binder, a disintegrating agent, alubricant, a corrigent, a solubilizing agent, a suspension aid, anemulsifying agent, a coating agent, a cyclodextrin, and/or a buffer.Although the dosage will vary depending on the symptoms, age and bodyweight of the patient, the nature and severity of the disorder to betreated or prevented, the route of administration and the form of thedrug, in general, a daily dosage of from 0.01 to 2000 mg of the compoundis recommended for an adult human patient, and this may be administeredin a single dose or in divided doses. The amount of active ingredientwhich can be combined with a carrier material to produce a single dosageform will generally be that amount of the compound which produces atherapeutic effect.

The precise time of administration and/or amount of the composition thatwill yield the most effective results in terms of efficacy of treatmentin a given patient will depend upon the activity, pharmacokinetics, andbioavailability of a particular compound, physiological condition of thepatient (including age, sex, disease type and stage, general physicalcondition, responsiveness to a given dosage, and type of medication),route of administration, etc. However, the above guidelines can be usedas the basis for fine-tuning the treatment, e.g., determining theoptimum time and/or amount of administration, which will require no morethan routine experimentation consisting of monitoring the subject andadjusting the dosage and/or timing.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose ligands, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition, or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial. Each carrier must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the patient. Some examples of materials which can serve aspharmaceutically acceptable carriers include: (1) sugars, such aslactose, glucose, and sucrose; (2) starches, such as corn starch, potatostarch, and substituted or unsubstituted β-cyclodextrin; (3) cellulose,and its derivatives, such as sodium carboxymethyl cellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6)gelatin; (7) talc; (8) excipients, such as cocoa butter and suppositorywaxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil,sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such aspropylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol,and polyethylene glycol; (12) esters, such as ethyl oleate and ethyllaurate; (13) agar; (14) buffering agents, such as magnesium hydroxideand aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17)isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20)phosphate buffer solutions; and (21) other non-toxic compatiblesubstances employed in pharmaceutical formulations. In certainembodiments, pharmaceutical compositions of the present invention arenon-pyrogenic, i.e., do not induce significant temperature elevationswhen administered to a patient.

The term “pharmaceutically acceptable salt” refers to the relativelynon-toxic, inorganic and organic acid addition salts of theinhibitor(s). These salts can be prepared in situ during the finalisolation and purification of the inhibitor(s), or by separatelyreacting a purified inhibitor(s) in its free base form with a suitableorganic or inorganic acid, and isolating the salt thus formed.Representative salts include the hydrobromide, hydrochloride, sulfate,bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate,stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate,maleate, fumarate, succinate, tartrate, naphthylate, mesylate,glucoheptonate, lactobionate, laurylsulphonate salts, and amino acidsalts, and the like. (See, for example, Berge et al. (1977)“Pharmaceutical Salts”, J. Pharm. Sci. 66: 1-19.)

In other cases, the inhibitors useful in the methods of the presentinvention may contain one or more acidic functional groups and, thus,are capable of forming pharmaceutically acceptable salts withpharmaceutically acceptable bases. The term “pharmaceutically acceptablesalts” in these instances refers to the relatively non-toxic inorganicand organic base addition salts of an inhibitor(s). These salts canlikewise be prepared in situ during the final isolation and purificationof the inhibitor(s), or by separately reacting the purified inhibitor(s)in its free acid form with a suitable base, such as the hydroxide,carbonate, or bicarbonate of a pharmaceutically acceptable metal cation,with ammonia, or with a pharmaceutically acceptable organic primary,secondary, or tertiary amine. Representative alkali or alkaline earthsalts include the lithium, sodium, potassium, calcium, magnesium, andaluminum salts, and the like. Representative organic amines useful forthe formation of base addition salts include ethylamine, diethylamine,ethylenediamine, ethanolamine, diethanolamine, piperazine, and the like(see, for example, Berge et al., supra).

Wetting agents, emulsifiers, and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring, and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like;(2) oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations suitable for oral administration may be in the form ofcapsules, cachets, pills, tablets, lozenges (using a flavored basis,usually sucrose and acacia or tragacanth), powders, granules, or as asolution or a suspension in an aqueous or non-aqueous liquid, or as anoil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup,or as pastilles (using an inert matrix, such as gelatin and glycerin, orsucrose and acacia) and/or as mouthwashes, and the like, each containinga predetermined amount of an inhibitor(s) as an active ingredient. Acomposition may also be administered as a bolus, electuary, or paste.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules, and the like), the active ingredient ismixed with one or more pharmaceutically acceptable carriers, such assodium citrate or dicalcium phosphate, and/or any of the following: (1)fillers or extenders, such as starches, cyclodextrins, lactose, sucrose,glucose, mannitol, and/or silicic acid; (2) binders, such as, forexample, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and/or acacia; (3) humectants, such as glycerol;(4) disintegrating agents, such as agar-agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, acetyl alcohol and glycerolmonostearate; (8) absorbents, such as kaolin and bentonite clay; (9)lubricants, such a talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and(10) coloring agents. In the case of capsules, tablets, and pills, thepharmaceutical compositions may also comprise buffering agents. Solidcompositions of a similar type may also be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugars, as well as high molecular weight polyethylene glycols, andthe like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered inhibitor(s)moistened with an inert liquid diluent.

Tablets, and other solid dosage forms, such as dragees, capsules, pills,and granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes, and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups,and elixirs. In addition to the active ingredient, the liquid dosageforms may contain inert diluents commonly used in the art, such as, forexample, water or other solvents, solubilizing agents, and emulsifierssuch as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, oils (in particular, cottonseed, groundnut, corn, germ, olive,castor, and sesame oils), glycerol, tetrahydrofuryl alcohol,polyethylene glycols, and fatty acid esters of sorbitan, and mixturesthereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming, and preservative agents.

Suspensions, in addition to the active inhibitor(s) may containsuspending agents as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

Formulations for rectal or vaginal administration may be presented as asuppository, which may be prepared by mixing one or more inhibitor(s)with one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, which is solid at room temperature, butliquid at body temperature and, therefore, will melt in the rectum orvaginal cavity and release the active agent.

Formulations which are suitable for vaginal administration also includepessaries, tampons, creams, gels, pastes, foams, or spray formulationscontaining such carriers as are known in the art to be appropriate.

Dosage forms for the topical or transdermal administration of aninhibitor(s) include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches, and inhalants. The active componentmay be mixed under sterile conditions with a pharmaceutically acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required.

The ointments, pastes, creams, and gels may contain, in addition toinhibitor(s), excipients, such as animal and vegetable fats, oils,waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc, andzinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to an inhibitor(s),excipients such as lactose, talc, silicic acid, aluminum hydroxide,calcium silicates, and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

The inhibitor(s) can be alternatively administered by aerosol. This isaccomplished by preparing an aqueous aerosol, liposomal preparation, orsolid particles containing the composition. A nonaqueous (e.g.,fluorocarbon propellant) suspension could be used. Sonic nebulizers arepreferred because they minimize exposing the agent to shear, which canresult in degradation of the compound.

Ordinarily, an aqueous aerosol is made by formulating an aqueoussolution or suspension of the agent together with conventionalpharmaceutically acceptable carriers and stabilizers. The carriers andstabilizers vary with the requirements of the particular composition,but typically include nonionic surfactants (Tweens, Pluronics, sorbitanesters, lecithin, Cremophors), pharmaceutically acceptable co-solventssuch as polyethylene glycol, innocuous proteins like serum albumin,oleic acid, amino acids such as glycine, buffers, salts, sugars, orsugar alcohols. Aerosols generally are prepared from isotonic solutions.

Transdermal patches have the added advantage of providing controlleddelivery of an inhibitor(s) to the body. Such dosage forms can be madeby dissolving or dispersing the agent in the proper medium. Absorptionenhancers can also be used to increase the flux of the inhibitor(s)across the skin. The rate of such flux can be controlled by eitherproviding a rate controlling membrane or dispersing the inhibitor(s) ina polymer matrix or gel.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more inhibitors(s) in combination withone or more pharmaceutically acceptable sterile aqueous or nonaqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,bacteriostats, solutes which render the formulation isotonic with theblood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents, and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include tonicity-adjusting agents, such as sugars, sodiumchloride, and the like into the compositions. In addition, prolongedabsorption of the injectable pharmaceutical form may be brought about bythe inclusion of agents which delay absorption such as aluminummonostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. For example, delayed absorption of a parenterallyadministered drug form is accomplished by dissolving or suspending thedrug in an oil vehicle.

Injectable depot forms are made by forming microcapsule matrices ofinhibitor(s) in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

The preparations of agents may be given orally, parenterally, topically,or rectally. They are, of course, given by forms suitable for eachadministration route. For example, they are administered in tablets orcapsule form, by injection, inhalation, eye lotion, ointment,suppository, infusion; topically by lotion or ointment; and rectally bysuppositories. Oral administration is preferred.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrasternal injection, and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a ligand, drug, or other materialother than directly into the central nervous system, such that it entersthe patient's system and thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

These inhibitors(s) may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracisternally, and topically, as by powders, ointmentsor drops, including buccally and sublingually.

Regardless of the route of administration selected, the inhibitor(s),which may be used in a suitable hydrated form, and/or the pharmaceuticalcompositions of the present invention, are formulated intopharmaceutically acceptable dosage forms by conventional methods knownto those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The concentration of a disclosed compound in a pharmaceuticallyacceptable mixture will vary depending on several factors, including thedosage of the compound to be administered, the pharmacokineticcharacteristics of the compound(s) employed, and the route ofadministration. In general, the compositions of this invention may beprovided in an aqueous solution containing about 0.1-10% w/v of acompound disclosed herein, among other substances, for parenteraladministration. Typical dose ranges are from about 0.01 to about 50mg/kg of body weight per day, given in 1-4 divided doses. Each divideddose may contain the same or different compounds of the invention. Thedosage will be an effective amount depending on several factorsincluding the overall health of a patient, and the formulation and routeof administration of the selected compound(s).

Another aspect of the invention provides a conjoint therapy wherein oneor more other therapeutic agents are administered with the proteasomeinhibitor. Such conjoint treatment may be achieved by way of thesimultaneous, sequential, or separate dosing of the individualcomponents of the treatment.

In certain embodiments, a compound of the invention is conjointlyadministered with one or more other proteasome inhibitor(s).

In certain embodiments, a compound of the invention is conjointlyadministered with a chemotherapeutic. Suitable chemotherapeutics mayinclude, natural products such as vinca alkaloids (e.g., vinblastine,vincristine, and vinorelbine); paclitaxel; epidipodophyllotoxins (e.g.,etoposide, teniposide); antibiotics (e.g., dactinomycin (actinomycin D)daunorubicin, doxorubicin and idarubicin); anthracyclines; mitoxantrone;bleomycins; plicamycin (mithramycin) and mitomycin; enzymes(L-asparaginase which systemically metabolizes L-asparagine and deprivescells which do not have the capacity to synthesize their ownasparagine); antiplatelet agents; antiproliferative/antimitoticalkylating agents such as nitrogen mustards (mechlorethamine,cyclophosphamide and analogs, melphalan, chlorambucil), ethyleniminesand methylmelamines (hexamethylmelamine and thiotepa); alkyl sulfonates(busulfan); nitrosoureas (carmustine (BCNU) and analogs, streptozocin);trazenes (e.g., dacarbazine (DTIC)); antiproliferative/antimitoticantimetabolites such as folic acid analogs (methotrexate), pyrimidineanalogs (fluorouracil, floxuridine, and cytarabine), purine analogs andrelated inhibitors (mercaptopurine, thioguanine, pentostatin and2-chlorodeoxyadenosine); aromatase inhibitors (anastrozole, exemestane,and letrozole), and platinum coordination complexes (cisplatin,carboplatin); procarbazine; hydroxyurea; mitotane; aminoglutethimide;hormones (e.g., estrogen); and hormone agonists such as leutinizinghormone releasing hormone (LHRH) agonists (e.g., goserelin, leuprolideand triptorelin).

In certain embodiments, the other therapeutic agent is an HDAC inhibitor(e.g., Trichostatin A, depsipeptide, apicidin, A-161906, scriptaid,PXD-101, CHAP, butyric acid, depudecin, oxamflatin, phenylbutyrate,valproic acid, SAHA (Vorinostat), MS275(N-(2-Aminophenyl)-4-[N-(pyridine-3-ylmethoxy-carbonyl)aminomethyl]benzamide),LAQ824/LBH589, CI994, or MGCD0103). In certain such embodiments, theother agent is SAHA (suberoylanilide hydroxamic acid).

In certain embodiments, the other therapeutic agent is a protein kinaseinhibitor (e.g., sorafenib, imatinib, dasatinib, sunitinib, pazopanib,and nilotinib). In certain such embodiments, the protein kinaseinhibitor is sorafenib.

In certain embodiments, the other chemotherapeutic agent is selectedfrom mechlorethamine, camptothecin, ifosfamide, tamoxifen, raloxifene,gemcitabine, navelbine, or any analog or derivative variant of theforegoing.

In certain embodiments, a compound of the invention is conjointlyadministered with a steroid. Suitable steroids may include, but are notlimited to, 21-acetoxypregnenolone, alclometasone, algestone,amcinonide, beclomethasone, betamethasone, budesonide, chloroprednisone,clobetasol, clocortolone, cloprednol, corticosterone, cortisone,cortivazol, deflazacort, desonide, desoximetasone, dexamethasone,diflorasone, diflucortolone, difuprednate, enoxolone, fluazacort,flucloronide, flumethasone, flunisolide, fluocinolone acetonide,fluocinonide, fluocortin butyl, fluocortolone, fluorometholone,fluperolone acetate, fluprednidene acetate, fluprednisolone,flurandrenolide, fluticasone propionate, formocortal, halcinonide,halobetasol propionate, halometasone, hydrocortisone, loteprednoletabonate, mazipredone, medrysone, meprednisone, methylprednisolone,mometasone furoate, paramethasone, prednicarbate, prednisolone,prednisolone 25-diethylaminoacetate, prednisolone sodium phosphate,prednisone, prednival, prednylidene, rimexolone, tixocortol,triamcinolone, triamcinolone acetonide, triamcinolone benetonide, ortriamcinolone hexacetonide, or a salt and/or derivative thereof.

In certain embodiments, a compound of the invention is conjointlyadministered with an immunotherapeutic agent. Suitable immunotherapeuticagents may include, but are not limited to, cyclosporine, thalidomide(or thalidomide analogs such as lenalidomide), or monoclonal antibodies.The monoclonal antibodies can be either naked or conjugated such asrituximab, tositumomab, alemtuzumab, epratuzumab, ibritumomab tiuxetan,gemtuzumab ozogamicin, bevacizumab, cetuximab, erlotinib or trastuzumab.

DEFINITIONS

The term “C_(x-y)alkyl” refers to substituted or unsubstituted saturatedhydrocarbon groups, including straight-chain alkyl and branched-chainalkyl groups that contain from x to y carbons in the chain, includinghaloalkyl groups such as trifluoromethyl and 2,2,2-trifluoroethyl, etc.C₀alkyl indicates a hydrogen where the group is in a terminal position,a bond if internal. The terms “C_(2-y)alkenyl” and “C_(2-y)alkynyl”refer to substituted or unsubstituted unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond respectively.

The term “alkoxy” refers to an alkyl group having an oxygen attachedthereto. Representative alkoxy groups include methoxy, ethoxy, propoxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl an ether is or resembles an alkoxy.

The term “C₁₋₆aralkyl”, as used herein, refers to a C₁₋₆alkyl groupsubstituted with an aryl group.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines and salts thereof, e.g., a moietythat can be represented by the general formulae:

wherein R⁹, R¹⁰ and R^(10′) each independently represent a hydrogen, analkyl, an alkenyl, —(CH₂)_(m)—R⁸, or R⁹ and R¹⁰ taken together with theN atom to which they are attached complete a heterocycle having from 4to 8 atoms in the ring structure; R⁸ represents an aryl, a cycloalkyl, acycloalkenyl, a heterocyclyl or a polycyclyl; and m is zero or aninteger from 1 to 8. In preferred embodiments, only one of R⁹ or R¹⁰ canbe a carbonyl, e.g., R⁹, R¹⁰, and the nitrogen together do not form animide. In even more preferred embodiments, R⁹ and R¹⁰ (and optionallyR^(10′)) each independently represent a hydrogen, an alkyl, an alkenyl,or —(CH₂)_(m)—R⁸. In certain embodiments, the amino group is basic,meaning the protonated form has a pK_(a)≧7.00.

The terms “amide” and “amido” are art-recognized as an amino-substitutedcarbonyl and includes a moiety that can be represented by the generalformula:

wherein R⁹, R¹⁰ are as defined above. Preferred embodiments of the amidewill not include imides which may be unstable.

The term “aryl” as used herein includes 5-, 6-, and 7-memberedsubstituted or unsubstituted single-ring aromatic groups in which eachatom of the ring is carbon. The term “aryl” also includes polycyclicring systems having two or more cyclic rings in which two or morecarbons are common to two adjoining rings wherein at least one of therings is aromatic, e.g., the other cyclic rings can be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline,and the like.

The terms “carbocycle” and “carbocyclyl”, as used herein, refer to anon-aromatic substituted or unsubstituted ring in which each atom of thering is carbon. The terms “carbocycle” and “carbocyclyl” also includepolycyclic ring systems having two or more cyclic rings in which two ormore carbons are common to two adjoining rings wherein at least one ofthe rings is carbocyclic, e.g., the other cyclic rings can becycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/orheterocyclyls.

The term “carbonyl” is art-recognized and includes such moieties as canbe represented by the general formula:

wherein X is a bond or represents an oxygen or a sulfur, and R¹¹represents a hydrogen, an alkyl, an alkenyl, —(CH₂)_(m)—R⁸ or apharmaceutically acceptable salt, R^(11′) represents a hydrogen, analkyl, an alkenyl or —(CH₂)_(m)—R⁸, where m and R⁸ are as defined above.Where X is an oxygen and R¹¹ or R^(11′) is not hydrogen, the formularepresents an “ester”. Where X is an oxygen, and R¹¹ is a hydrogen, theformula represents a “carboxylic acid”.

The terms “heteroaryl” includes substituted or unsubstituted aromatic 5-to 7-membered ring structures, more preferably 5- to 6-membered rings,whose ring structures include one to four heteroatoms. The term“heteroaryl” also includes polycyclic ring systems having two or morecyclic rings in which two or more carbons are common to two adjoiningrings wherein at least one of the rings is heteroaromatic, e.g., theother cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls,aryls, heteroaryls, and/or heterocyclyls. Heteroaryl groups include, forexample, pyrrole, furan, thiophene, imidazole, oxazole, thiazole,triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, andthe like.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen,phosphorus, and sulfur.

The terms “heterocyclyl” or “heterocyclic group” refer to substituted orunsubstituted non-aromatic 3- to 10-membered ring structures, morepreferably 3- to 7-membered rings, whose ring structures include one tofour heteroatoms. The term terms “heterocyclyl” or “heterocyclic group”also include polycyclic ring systems having two or more cyclic rings inwhich two or more carbons are common to two adjoining rings wherein atleast one of the rings is heterocyclic, e.g., the other cyclic rings canbe cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/orheterocyclyls. Heterocyclyl groups include, for example, piperidine,piperazine, pyrrolidine, morpholine, lactones, lactams, and the like.

The term “C₁₋₆hydroxyalkyl” refers to a C₁₋₆alkyl group substituted witha hydroxy group.

The terms “polycyclyl” or “polycyclic” refer to two or more rings (e.g.,cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/orheterocyclyls) in which two or more carbons are common to two adjoiningrings, e.g., the rings are “fused rings”. Each of the rings of thepolycycle can be substituted or unsubstituted.

The term “proteasome” as used herein is meant to include immuno- andconstitutive proteasomes.

The term “substantially pure” as used herein, refers to a crystallinepolymorph that is greater than 90% pure, meaning that contains less than10% of any other compound, including the corresponding amorphouscompound. Preferably, the crystalline polymorph is greater than 95%pure, or even greater than 98% pure.

The term “substituted” refers to moieties having substituents replacinga hydrogen on one or more carbons of the backbone. It will be understoodthat “substitution” or “substituted with” includes the implicit provisothat such substitution is in accordance with permitted valence of thesubstituted atom and the substituent, and that the substitution resultsin a stable compound, e.g., which does not spontaneously undergotransformation such as by rearrangement, cyclization, elimination, etc.As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and non-aromaticsubstituents of organic compounds. The permissible substituents can beone or more and the same or different for appropriate organic compounds.For purposes of this invention, the heteroatoms such as nitrogen mayhave hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valences of theheteroatoms. Substituents can include, for example, a halogen, ahydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl,or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or athioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, aphosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro,an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, asulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or anaromatic or heteroaromatic moiety. It will be understood by thoseskilled in the art that the moieties substituted on the hydrocarbonchain can themselves be substituted, if appropriate.

A “therapeutically effective amount” of a compound with respect to thesubject method of treatment, refers to an amount of the compound(s) in apreparation which, when administered as part of a desired dosage regimen(to a mammal, preferably a human) alleviates a symptom, ameliorates acondition, or slows the onset of disease conditions according toclinically acceptable standards for the disorder or condition to betreated or the cosmetic purpose, e.g., at a reasonable benefit/riskratio applicable to any medical treatment.

As used herein, the term “treating” or “treatment” includes reversing,reducing, or arresting the symptoms, clinical signs, and underlyingpathology of a condition in manner to improve or stabilize a subject'scondition.

EXEMPLIFICATION Example 1

Amorphous Compound 1 (90.8% purity by HPLC, 6.56 g) prepared accordingto U.S. patent application Ser. No. 11/820,490, was dissolved in toluene(129 mL) in a oil bath at 60° C., the solution was cooled to ambienttemperature, and the mixture was stirred for three hours. The resultingcrystals were filtered, washed with toluene and dried under vacuum for12 hours at 22° C. to provide a toluene solvate of Compound 1 (99.4%purity by HPLC, 4.63 g).

The characteristic DSC curve of the sample as shown in FIG. 1 wasrecorded on a TA Instruments Differential Scanning calorimeter 2920 at aheating rate of 10° C./minute.

The characteristic X-ray powder pattern as shown in FIG. 2 was recordedon a Shimadzu XRD-6000 under Cu Kα radiation [voltage and current set at40 kV and 40 mA; divergence and scattering slits set at 1° and receivingslit set at 0.15 mm; NaI scintillation detector used for diffractedradiation; a θ-2θ continuous scan at 3°/min (0.4 sec/0.02° step) from2.5 to 40° 2θ was used; samples were placed in an aluminum holder withsilicon insert; and data collected and analyzed with XRD-6100/7000v.5.0].

Thermogravimetric analyses were performed with a TA Instruments 2950 ata heating rate of 10° C./minute.

Example 2

Amorphous Compound 1 (221 g) prepared according to U.S. patentapplication Ser. No. 11/820,490, was dissolved in 1:1 toluene-methyltert-butyl ether (4.2 L), seeded with a crystalline toluene solvate ofCompound 1, and allowed to crystallize at 15° C. to 25° C. over 24hours. The crystals were filtered, washed with heptanes and dried undervacuum for 24 hours at 40° C. to provide a crystalline toluene solvateof Compound 1 (97.0% purity by HPLC, 115 g).

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thecompounds and methods of use thereof described herein. Such equivalentsare considered to be within the scope of this invention and are coveredby the following claims.

All of the above-cited references and publications are herebyincorporated by reference.

I claim:
 1. A method for preparing a crystalline compound of Formula(II)

wherein the crystalline compound has 2θ values 3.62±0.20; 6.52±0.20;7.20±0.20; 8.66±0.20; 10.04±0.20; 10.82±0.20; 12.98±0.20; 14.42±0.20;16.46±0.20; 17.32±0.20; 17.98±0.20; 18.02±0.20; 18.88±0.20; 19.06±0.20;19.56±0.20; 19.88±0.20; 20.40±0.20; 21.36±0.20; 22.16±0.20; 23.20±0.20;25.40±0.20; and 28.82±0.20, comprising (i) preparing a solution of acompound of Formula (II) in an organic solvent; (ii) bringing thesolution to supersaturation to cause formation of crystals; and (iii)isolating the crystals.
 2. The method of claim 1, wherein the organicsolvent is selected from acetonitrile, acetone, diethyl ether,diisopropyl ether, ethyl acetate, heptanes, isopropyl acetate, methanol,methyl tert-butyl ether, tetrahydrofuran, toluene, or any combinationthereof.
 3. The method of claim 2, wherein the organic solvent isselected from toluene, methyl tert-butyl ether, heptanes, or acombination thereof.
 4. The method of claim 1, wherein bringing thesolution to supersaturation comprises addition of an anti-solvent,allowing the solution to cool, reducing the volume of the solution, orany combination thereof.
 5. The method of claim 4, wherein bringing thesolution to supersaturation comprises cooling the solution to ambienttemperature and reducing the volume of the solution.
 6. The method ofclaim 1, further comprising washing the crystals.
 7. The method of claim6, wherein the washing comprises washing with a liquid selected fromanti-solvent, acetonitrile, acetone, diethyl ether, diisopropyl ether,ethanol, ethyl acetate, heptanes, isopropyl acetate, methanol, methyltert-butyl ether, tetrahydrofuran, toluene, or any combination thereof,or a combination thereof.
 8. The method of claim 6, wherein washingcomprises washing with a combination toluene and methyl tert-butylether.
 9. The method of claim 7, wherein washing comprises washing withtoluene.
 10. The method of claim 1, wherein isolating the crystalscomprises filtering the crystals.
 11. The method of claim 1, furthercomprising drying the crystals under reduced pressure.
 12. The method ofclaim 1, wherein the crystalline compound of Formula (II) is a solvate.13. The method of claim 12, wherein the crystalline compound of Formula(II) is a toluene solvate.
 14. A crystalline compound having a structureof Formula (II)

wherein the crystalline compound has 2θ values 3.62±0.20; 6.52±0.20;7.20±0.20; 8.66±0.20; 10.04±0.20; 10.82±0.20; 12.98±0.20; 14.42±0.20;16.46±0.20; 17.32±0.20; 17.98±0.20; 18.02±0.20; 18.88±0.20; 19.06±0.20;19.56±0.20; 19.88±0.20; 20.40±0.20; 21.36±0.20; 22.16±0.20; 23.20±0.20;25.40±0.20; and 28.82±0.20.
 15. The crystalline compound of claim 14,having a DSC thermogram as shown in FIG.
 1. 16. The crystalline compoundof claim 14, having an XRPD pattern as shown in FIG.
 2. 17. Thecrystalline compound of claim 14, which is a solvate.
 18. Thecrystalline compound of claim 17, which is a toluene solvate.
 19. Amethod for treating a disease or condition selected from animmune-related disease, cancer, inflammation, infection, proliferativedisease, or neurodegenerative disease, comprising administering acrystalline compound of claim
 14. 20. The method of claim 19, whereinthe disease or condition is an immune-related disease.
 21. The method ofclaim 20, wherein the immune-related disease is selected from autoimmunedisease, Crohn's disease, diabetes, lupus, multiple sclerosis, myositis,psoriasis, rheumatoid arthritis and ulcerative colitis.
 22. The methodof claim 19, wherein the disease or condition is cancer.
 23. The methodof claim 22, wherein the cancer is selected from leukemia, lymphoma andmultiple myeloma.
 24. The method of claim 19, wherein the disease orcondition is inflammation.
 25. The method of claim 19, wherein thedisease or condition is an infection.
 26. The method of claim 19,wherein the disease or condition is a proliferative disease.
 27. Themethod of claim 19, wherein the disease or condition is aneurodegenerative disease.